Download 28-lymphoma-and-lymphoproliferative-feb-2014

10/6/2016
LYMPHOMA &
LYMPHOPROLIFERATIVE DX
BACKGROUND
Lymphomas and LPD’s are neoplasms of T, B, or NK
lymphoid cells and their precursors
Although having different characteristics from their
KAREN FERREIRA MS,MLS (ASCP)
ASSOCIATE SCIENTIFIC DIRECTORHEMATOLOGY
LIFESPAN ACADEMIC MEDICAL CTR
10/19/2016
normal counterparts, the neoplastic cells of many
lymphoid diseases have the features of lymphoid cells at
a particular stage of differentiation
Neoplastic lymphoid cells can have the characteristics of
lymphocytes that normally reside in a particular organ or
tissue
Neoplastic lymphocytes tend to home to the tissues and
specific locations where their normal counterparts reside
Molecular Basis of Lymphoid Neoplasm
Lymphoid Neoplasm
Lymphoid neoplasms arise as a result of a series of
mutations in a single lymphoid cell
This is usually in a committed lymphoid cell (T, B,
NK)
Mutations are variable but usually involve oncogenes
and the loss of tumor suppressor genes
Standard cytogenetic analysis can diagnose these
translocations, deletions or inversions
An understanding of the normal immune system is
necessary to understand the nature of
LPD’s/lymphoma
Normal Immune System
Organs
Bone marrow
Lymph nodes
Spleen
Thymus
Liver
GI tract
Mucosal-associated lymphoid tissue (MALT)
Immune System
The components of the immune system are connected by
lymphatic channels and the blood stream
Other cells involved
Neutrophils (phagocytes)
Monocytes/macrophages (APC’s)
3. Dendritic cells (APC’s)
Innate immunity-does not require prior antigen
exposure-immediate response-phagocytic cells, NK
cells, and plasma proteins of the complement system
Adaptive immunity-response to antigen exposurespecificity and memory-delayed response
1.
2.
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Lymphoid Tissue
Organs of the Immune System
Primary lymphoid organs
1.
Bone marrow
2. Thymus
Secondary lymphoid organs
Lymph nodes
2. Spleen
3. Other peripheral lymphoid tissue
1.
Normal Lymph Node Structue
1.
Cortex
Primary follicles are composed of B-cells and dendritic
cells
2. Upon antigen exposure, proliferation and
differentiation of B-cells cause the primary follicle to
develop into a secondary follicle comprising a germinal
center surrounded by a mantle zone of small Blymphocytes
3. Outside the mantle zone many follicles have a marginal
zone also comprised of B-cells
4. The network of dendritic cells in the germinal center
presents antigen to B-cells
1.
Cortex
2. Paracortex
3. Medulla
Lymph Node Structure
Paracortex
1.
Occupied by T-cells
2. Surrounds the primary and secondary follicles
3. Abundant dendritic cells
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Medulla
Lymph Node Structure
The center of the lymph node-composed of
medullary cords and sinuses
2. Medulary cords T-cells
B-cells
Plasma cells
Macrophages
1.
Lymph Node Structure
Lymph Node
Lymph-derived from interstitial fluid and containing
a variable number of lymphocytes, is brought to the
lymph nodes by afferent lymphatics and is
transported from the lymph nodes by an efferent
lymphatic system
Lymphs are also brought to the nodes by its arterial
supply
Lymph Node
Lymphocytes recirculate from the nodes or other
lymphoid tissue through the lymphatics and the
blood stream back to lymphatic tissue
Homing of lymphs to tissues similar to those from
which they originated (skin, GI) is usual
Normal B-cell Development
A hematopoietic stem cell in the bone marrow gives rise
to a B-cell precursor and then to a naïve B-cell, which
migrates either to secondary lymphoid tissue such as a
lymph node primary follicle or medulla
If the B-cell is presented with antigen by a dendritic cell
or macrophage further development occurs
A naïve (IgM or IgD) B-cell in the primary follicle
responds to antigen by class switching and migration to
the mantle zone
The mantle zone B-cell then migrates back into the
germinal center and transforms to a centroblast and
then a centrocyte within what is now a secondary follicle
containing a germinal center
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B-cell development cont.
Benign Reactive Lymph Node
These germinal center cells undergo a somatic
hypermutation before migrating to the marginal
zone and then the blood stream
Post-germinal center B-cells become memory cells in
blood or tissues or plasma cells in tissues
Reactive hyperplasia
Reactive Lymph Node
Normal T-cell Development
Positive selection
Hematopoietic stem cells in the bone marrow give
If cortical thymocytes recognize a specific foreign
rise to T-cell precursors, which enter the thymic
medulla and then migrate from the medulla to the
cortex
Cortical thymocytes undergo positive selection
peptide presented in a MHC I or II context by thymic
epithelial cells they will survive-if not they undergo
apoptosis
Surviving cells develop into either CD4+ or CD8+
cells which migrate to the medulla
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Thymus
T & B cell gene rearrangements
T and B cell precursors have germline genes that are unusual in
that they are divided into segments
These must be assembled into functional genes by a process of
deletion and rearrangement of gene segments to form the genes
that encode the various immunoglobulin molecules (IgH, Igk,
Igl) and similarly, the T-cell receptor genes (TCR’s α, β, γ, δ)
Once a light chain gene has been effectively rearranged,
immunoglobulin is expressed on the surface
There is a greater degree of genetic rearrangement occuring in Bcells that make B cell lymphomas far more common than T-cells
T cell gene rearrangements
NK cells
Most mature T-cells have a surface membrane
NK cells are part of innate immunity
complex composed of α and β chains of the T-cell
receptor together with CD3 and either CD4 or CD8
which recognize specific peptides in an MHC context
CD8 positive-MHC class I
CD4 positive-MHC class II
Do not undergo any gene rearrangements
Immunophenotype
LYMPHOID DIFFERENTIATION
The genetic rearrangement that occurs in B and T
cells is paralleled by alterations in expansion of
surface membrane and cytoplasmic antigens
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Lymphoma/LPD
There is a putative relationship
between the normal stages of Bcell and T-lineage differentiation,
and B- and T- cell lineage
neoplasms
Lymphoma
Lymphoproliferative Disorders
Peripheral Blood
Bone Marrow
Lymphoma/Lymphoproliferative
Lymph Nodes
Precursor lymphoid neoplasms
Secondary Lymphoid Organs
Mature B-cell neoplasms
Mature T-cell neoplasms
NK-neoplasms
Hodgkin lymphoma
Precursor Lymphoid Neoplasms
Mature B-cell neoplasms
B lymphoblastic leukemia/lymphoma
Chronic lymphocytic leukemia/lymphoma
T lymphoblastic leukemia/lymphoma
Splenic marginal zone lymphoma
Hairy cell leukemia
Follicular lymphoma
Burkitt’s
Mantle cell lymphoma
Diffuse large B-cell lymphoma
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DEFINITION-LEUKEMIA
A malignant disease of hematopoietic tissue,
characterized by replacement of normal bone
marrow elements with abnormal (neoplastic) blood
cells.
Acute leukemia-proliferation of primitive cells (rapid
course)
Chronic leukemia-proliferation of mature, welldifferentiated cells (slow course)
CLL/SLL
CLL/SLL
Definition
A neoplasm composed of monomorphic small, round
lymphocytes in the PB, BM, spleen and LN’s
In the absence of extramedullary tissue involvement
there must be > 5 x 10 9/l monoclonal lymphocytes
with CLL phenotype
CLL-Epidemiology
Lymphocytosis must be present for >3 months
Most common leukemia of adults in Western world
The term SLL is used for cases with tissue
2-6 cases per 100,000 persons/year increasing with
morphology and immunophenotype of CLL
age to 12.8/100,000 at age 65
6.7% of all non-Hodgkin lymphoma
CLL-sites of involvement
Morphology-SLL
PB, BM
Lymph nodes
Liver
Spleen
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Case 1
Morphology
59 yr old male presents in ER
WBC=171.9 x 109/l
HGB=5.7g/dl
Plt=33,000/cumm
Lymphocytes=88%
Absolute lymphocyte count=151.3 x 109/l
Flow Cytometry
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Flow Cytometry
Flow Cytometry
Flow Cytometry
ZAP-70
Zap-70 is a prognostic marker in CLL
●low levels of Zap-70→mutated IgVH genes=good
●high levels of Zap-70→unmutated IgVH genes=bad
Correlate ZAP-70 with CD38 Expression
Case 1-Diagnosis
B-CHRONIC LYMPHOCYTIC LEUKEMIA
-absolute lymphocytosis
-smudge cells on PB smear may require albumin prep
-most common “chronic” leukemia in Western world
IMMUNOPHENOTYPE
B-Cells (CD19+,CD20+DIM) that co-express CD5
CD23+
Must prove CLONALITY
MONOCLONAL = LIGHT CHAIN RESTRICTION
Dim kappa or lambda
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Hairy Cell Leukemia
Indolent neoplasm of small mature B lymphoid cells
with oval nuclei and abundant cytoplasm with
“hairy” projections
Involves PB, BM, and splenic red pulp
Patients present with weakness and fatigue, left
upper quadrant pain, fever, and bleeding
HCL
Splenomegaly and pancytopenia with only a few
circulating neoplastic cells is common
Only 2% of all lymphoid neoplasms
HAIRY CELL LEUKEMIA
Cytochemistry
♂:♀ 7:1
TARTRATE RESISTENT ACID PHOSPHATASE +
Pancytopenia
Blood films are incubated in AS-BI phosphoric acid
Splenomegaly-diffuse red pulp infiltrate
Bone marrow dry tap due to reticulin fibrosis. Hairy
cells produce basic fibroblast growth factor (bFGF)
which increases reticulin in BM
TRAP
and freshly diazotized fast garnet GBC.
Duplicate slides are incubated with the substrate +
L(+) tartrate
TRAP Positive Hairy Cells
Napthol AS-BI released by enzymatic hydrolysis
couples immediately with fast garnet GBC forming
insoluble maroon dye deposits at sites of activity
The acid phosphatase of most normal and malignant
cells is inhibited by tartrate but the acid phosphatase
of hairy cells (isoenzyme 5) is resistant to tartrate
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IMMUNOPHENOTYPE
Follicular Lymphoma
B-cells CD19+, CD20bright
Neoplasm comprised of follicle center (germinal
CD103**
center) B-cells which have a follicle pattern on the
LN section
Accounts for 20% of all lymphomas
Predominantly involves lymph nodes
But also invades BM, PB, and spleen
CD25
CD11c
BRIGHT MONOCLONAL sIg+
Follicular Lymphoma
Lymph Node Architecture -FL
Most patients have widespread disease at the time of
dx-including peripheral and central (abdominal and
thoracic) lymphadenopathy and splenomegaly
The bone marrow is involved in 40-70%
Only 1/3 of patients are in Stage 1 or II at dx
Leukemic Phase of FL
PB involvement of FL
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Immunophenotype
Cytogenetics
CD19+
Immunoglobulin light and heavy chains are
CD20+
rearranged; variable region genes show extensive
and ongoing somatic hypermutation
T(14;18)
Bcl-2 gene rearrangements
CD10+
Bcl-2+
Bcl-6+
Monoclonal sIg light chain+
Prognosis
Dependant on the extent of the disease at diagnosis
BURKITT’S LEUKEMIA
Endemic form is the most common type of childhood
cancer in Africa where it is associated with EBV
Sporadic form found in Western world
300 cases in the USA/year
Male:female 2-3:1
T(8;14)
T(8:14)
In 90% of cases of Burkitt’s a
reciprocal translocation has
moved the proto-oncogene cmyc from chromosome 8 to a
location close to the
enhancers of HC gene on
chromosome14
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Burkitt’s
Uncontrolled mitosis results in a clone.
Other translocations include:
t(2;8) kappa
t(8;22) lambda
Touch prep Burkitt’s lymphoma
Burkitt’s starry sky
Starry night
Starry sky
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Immunophenotype
Case 5
B-cells (CD19+,CD20+)
70 year old male
CD10 positive
WBC= 17.9 x 109/l
CD34 never positive
HGB= 15.7g/dl
TdT never positive
PLt= 174,000/cumm
Always with sIgM and monoclonal light chain
71% lymphs
restriction
Morphology- lymphs are basophilic and possess a
single prominent nucleolus
PB Morphology (10x)
Flow Cytometry
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Flow Cytometry
Flow Cytometry
Molecular Biology
Case 5- Diagnosis
Molecular studies are positive for TCR beta and TCR
gamma gene rearrangement
Mature Clonal T-Cell Lymphocytosis Consistent with
T-Cell Prolymphocytic Leukemia
Flow cytometry of PB lymphs shows 92% T-cells
expressing CD2,CD3,CD4,CD5,CD7,TCRαβ
Recommend HTLV-1, cytogenetic studies and bone
marrow evaluation as clinically indicated
Infection and Cancer
Infection contributes to 15-20% of all human
cancers
Infection can cause long-term inflammation,
suppress a person’s immune system, or directly
affect a cell’s DNA
Bacterial, viral, and parasitic infections have all
been linked to cancer
Viral Infection and Cancer
Human papilloma virus-cervical cancer
Hepatitis B and C- liver cancer
Human herpes virus 8 (HHV-8)- Kaposi’s sarcoma
Feline leukemia virus
Simian virus 40-mesothelioma in monkeys
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Viral Infection and Leukemia
EBV endemic in Africa and associated with Burkitts
lymphoma (infects and immortalizes B-cells)
Human T-lymphotropic virus endemic in Japan and
some parts of the Caribbean basin is associated with
Adult T-cell Leukemia Lymphoma (infects and
immortalizes CD4+ T-cells)
Viral Infection and Cancer
Viruses encode proteins that that reprogram host
cellular signaling pathways that control proliferation,
differentiation, cell death, genomic integrity, and
recognition by the immune system.
Background
HTLV-1 is an exogenous retrovirus that infects 20-30
million people worldwide
Causes ATLL and HTLV-1 associated
myelopathy/tropical paraparesis (CNS disease)
Infects CD4+ T-cells
First human retrovirus discovered (1977)
Viral Properties
Deltavirus genera of the Orthoretrovirinae
subfamily
Spherical, enveloped retrovirus with a diameter of
80-120nm
The envelope surrounds a capsid that contains two
identical copies of positive-strand genome inside an
electron dense core.
Tax- a trancriptional activator
Tax
Role in viral replication
Promotes accelerated cell cycle
Transformation of infected cell
Proliferation
Activates and/or deactivates transcription from viral
Obliteration of programmed cell death
promoter
Modulates transcription
Affecting cell growth and differentiation, cell cycle
control and DNA repair
Known to deregulate expression of more than 100
genes
Multiple Tax functions cooperate together for
malignant transformation of HTLV-1 infected cells
Induces expression of IL-1, which stimulates
osteoclast activity
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Tax
Epidemiology
10-20 million HTLV-1 carriers worldwide
Endemic in Japan (15-30%) of the population. (1.2
million people infected)
Caribbean basin (3-6%)
South America (2%)
US blood donors 0.025% HTLV-1 seropositivity.
Seropositivity among US prostitutes and IV drug
abusers→7-49%
Transmission – 3 routes
Mother to infant – mostly breast milk but also
intrauterine infection
2. Sexual transmission- the rate of transmission
between husband and wife over a 10 year period is
60.8% from husband to wife and 0.4% from wife to
husband
3. Parenteral transmission – blood transfusion or IV
drug use
1.
Adult T-Cell Leukemia Lymphoma
Generalized LN, PB, and skin involvement by
pleomorphic tumor cells, ranging from small to large
in size with characteristic “flower” shape nuclei
Patients present with lytic bone lesions and
hypercalcemia
Rapidly progressive course- short survival
ATLL- 4 clinical presentations
Studies indicate a long latency period for HTLV-1 and
lifetime ATLL risk of less than 5% in infected
individuals
Acute
Lymphomatous
Chronic
Smouldering
ATLL- Acute
55-60% of cases
Severe PB lymphocytosis with numerous “flower”
cells
Hypercalcemia
Hepatoslenomegaly
Elevated serum LDH
Median survival is 6 months despite multiagent
chemotherapy
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ATLL – “Flower” Cells
ATLL - Chronic
15-20% of cases
Normal to slightly elevated lymphocyte count with
some circulating “flower” cells
Median survival is 2 years, but most patients die
within 5 years due to progression to acute or
lymphomatous phases
Diagnosis
Documentation of HTLV-1 infection by serologic
analysis or detection of clonal integration of HTLV-1
genome into neoplastic cells by molecular techniques
Vigilant morphologic analysis of PB lymphocyte
morphology
Documentation of specific ATLL immunophenotype
(flow cytometry or immunohistochemistry)
Case Study 6
ATLL- Immunophenotype
CD2+, CD3+ (dim), CD4+, *CD25+ (IL-2 receptor),
aberrant loss of pan T-cell marker CD7
*When a T-cell is activated, it secretes IL-2, at the
same time it upregulates the IL-2 receptor on its’
surface. The secreted IL-2 the binds to the
upregulated IL-2 receptor and results in cell division
PB Wright’s stain x1000
60 yo male with weakness referred to Heme/Onc by
PCP
Calcium 15.5 mg/dL –hypercalcemia
Small palpable left cervical LN
Liver enzymes increased
WBC 90.0 x 109/L with 6% blasts,
8% atypical lymphocytes, 63% lymphocytes
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PB
PB
PB
PB
PB
PB
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BM asp Wright’s stain x1000
BM bx H&E stain x200
BM bx
Immunophenotyping
Flow Cytometry
Flow Cytometry
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Case 7
Flow Cytometry
CD25 positivity is
hallmark for ATLL
57 year old female
Post kidney transplant
WBC= 6.9 x 109/l
HGB=7.5g/dl
Plt=386,000/cumm
Normal diff
Pleural Effusion- nucleated cells=1340/cumm
Pleural Fluid (20x)
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Viral load
The pleural fluid has 2 million copies/ml of EBV
Molecular Biology
Heavy chain gene rearrangement is detected by PCR
compared with 277,000 in the blood
Case 7- Diagnosis
EBV positive post transplant lymphoproliferative
malignancy with morphologic and immunophenotypic
evidence of plasma cell differentiation
Basophilic cells, large and small. CD45dim,
CD19,CD20,CD22,CD52,CD30 are negative. T-cell
markers are negative. CD38, CD138, and CD56 are
strongly +. sIG light chains are neg, but there is cIg
kappa restriction.
CBC Interpretation
RBC 3.45x10^12/L
HGB 11.4g/dL
HCT 34.1%
0.6x10^9/L
MCV 99.0fL
2.2x10^9/L
MCH 33.1pg
0.1x10^9/L
MCHC 33.5g/dL
RDW 16.9%
Platelets 62x10^9/L
WBC 3.0x10^9/L
Absoulute Values:
Neutrophil
Lymphocytes
Monocytes
Eosinophils 0x10^9/L
Basophils 0x10^9/L
Case #3
Patient History
A 60 year old female presents to her
physician with noticeably easy bruising and
profound fatigue.
History of Plavix use in the past. Not currently
taking any medication besides baby aspirin.
Past Medical History: Remarkable for
arthritis, hypertension and hypothyroidism.
A year ago, she had a normal white count,
normal hemoglobin and platelet count
99,000, slightly below normal value.
Peripheral Blood Review
Pancytopenia with an absolute
monocytopenia as well as neutropenia.
Absolute lymphocyte number is within
normal range however, a large subset show
medium sized nucleoli and abundant
cytoplasm with numerous hair-like
projections.
Rare nucleated RBC seen on scanning.
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Peripheral Blood Morphology
Peripheral Blood Morphology
Peripheral Blood Morphology
Peripheral Blood Morphology
Bone Marrow Aspirate Smear
Bone Marrow Sinusoidal Morphology
No cellular bone marrow spicules for
evaluation- poor aspirate sample or dry tap?
Predominant cell type in sinusoidal blood:
Lymphocytes; most being small to
medium sized with slightly larger nuclei and
occasional nucleoli.
Significantly decreased myeloid lineage.
Erythrocyte and megakaryocyte series are
essentially absent in the sinusoidal blood.
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Bone Marrow Sinusoidal Morphology
Bone Marrow Sinusoidal Morphology
Cytochemical Stains
Tartrate Resistant Acid Phosphatase stain
Tartrate Resistant Acid Phosphatase stain
(TRAP): Acid Phosphatase (isoenzyme 5) is
resistant to tartrate. A specific subset of
lymphocytes as well as histiocytes contain this
acid phosphatase compound and are therefore
resistant to inhibition, and will demonstrate
positivity.
Gaucher Disease is another clinical condition
in which TRAP staining is positive.
Our TRAP stain revealed that the subset of
lymphocytes with the hair-like projections are
positive for TRAP staining.
Tartrate Resistant Acid Phosphatase stain
Bone Marrow Biopsy
The decalcified bone marrow biopsy consists
of a core of trabecular bone that measured
0.8cm in length.
The cellularity ranges from 50-100%, overall
approximatley 70% cellular.
Architecture of the bone marrow is
predominantly effaced by lymphocytes with
abundant cytoplasm giving a “fried egg”
appearance.
The lymphocytes replace >75% of the marrow,
leaving behind only pockets of hematopoietic
marrow.
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Immunohistochemical Stains
Core biopsy
CD20
PAX-5
Annexin A1
All the above stains highlight a the specific
lymphocyte population in question with near
effacement of the marrow.
CD20
CD3
FLOW CYTOMETRY HISTOGRAMS-Gating
Monoclonal Antibodies-T and B-cells
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ISOLATING B-CELLS
ISOLATING B-CELLS
B-CELLS
SURFACE IG LIGHT CHAINS
Over View
Slow-growing lymphoid malignancy.
Future procedures include a CT of the chest,
abdomen and pelvis to assess for adenopathy,
Treatment plan: 1-week treatment with
Cladribine as a 2-hour infusion and then 4
weeks later give 8 consecutive treatments with
Rituximab.
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